The role ofthe endothelium and its relation to hemodynamics is a prominent candidate for atherosusceptibility in regions of complex disturbed flow. Endothelium at susceptible sites in undiseased arteries express a distinct set of balances of pro-pathological and protective pathway enhancement that define the susceptible endothelial phenotype. In Project 5 we propose (a) that hypercholesterolemia (HC) will change the phenotype balance of susceptible sites in different arteries with distinct spatial and temporal footprints, and (b) in complementary in vitro studies, that hemodynamic flow characteristics are epigenetic regulators of phenotype balance. We propose unbiased genomic analyses and focused mechanistic investigations of a primary perturbed pathway, endoplasmic reticulum (ER) stress, as complementary approaches to phenotype transition in vivo and in vitro. In Aim 1, endothelial phenotypes of 11 arterial regions will be mapped spatially and temporally during 0-6 months of HC in adult swine in an ongoing collaboration with the FDA Center for Devices and Radiological Health in Laurel, MD. Particular focus will be upon endothelial endoplasmic reticulum (ER) stress/unfolded protein response (UPR) pathways that we have very recently identified as characteristic of athero-susceptibility in normal animals in vivo. Phenotyping of small numbers of endothelial cells (EC) including those overlying developing lesions wili be conducted following laser capture microscopy, linear mRNA amplification and microarray, and immunostaining of targeted proteins identified in the transcript studies. In Aim 2, the contributions of hemodynamic forces and lipid environment to endothelial phenotype transition will be measured in vitro in EC exposed to arterial flow waveforms. The influence of controlled flow characteristics on mRNA transcript profiles and miRNA differential expression - with special interest in miR10a/10b regulation will be investigated. In Aim 3, focus will be directed to endothelial ERstress and UPR mechanisms at the protein level both in vivo and in vitro. The proposal addresses at the discovery and mechanistic levels how spatially-defined as a fnction of endothelium transitions from a propathological imbalance to a pathological phenotype as a function of its lipid and hemodynamic environment.